A method for preparing a reconstituted tobacco leaf based on microbial fermentation, a reconstituted tobacco leaf, and a heated cigarette

Abstract

This application discloses a method for preparing reconstituted tobacco leaves based on microbial fermentation, as well as reconstituted tobacco leaves and heated cigarettes. The method includes fermentation using microbial strains, centrifugation to obtain fermentation supernatant and microbial cells, suspension of the microbial cells to obtain an inoculum solution; fermentation of tobacco stem material using the inoculum solution to obtain fermentation products; recycling of the fermentation products to obtain a suspension, followed by heating to inactivate the microorganisms and enzymes in the suspension to obtain tobacco stem fiber material; mixing the tobacco stem fiber with tobacco dust for pulping, followed by papermaking, pressing, and drying to obtain a dried sheet base; concentrating the fermentation supernatant to obtain a coating solution, coating the coating solution onto the dried sheet base, and drying to obtain reconstituted tobacco leaves. This method achieves comprehensive modification of tobacco stem fibers through "dual-purpose microbial treatment and integrated fermentation-nanotechnology," allowing the microbially treated fibers to be directly used in reconstituted tobacco leaf papermaking, significantly improving the sheet base strength and antibacterial properties.

Description

Technical Field

[0001] This invention belongs to the field of tobacco processing technology, specifically relating to a method for preparing reconstituted tobacco leaves based on microbial fermentation, the reconstituted tobacco leaves, and heated cigarettes. Background Technology

[0002] Tobacco stems, consisting of the coarse, stiff veins of tobacco leaves, account for approximately 25-30% of the total weight of tobacco leaves. They are one of the main raw materials for reconstituted tobacco in papermaking, typically processed from tobacco byproducts such as stems and broken leaves through processes including extraction, pulping, papermaking, and coating. However, tobacco stems contain a high lignin content (6-10%), resulting in coarse, stiff fibers with poor flexibility. Direct use in papermaking leads to low strength and poor bulk of the resulting tobacco sheets, severely impacting the processing performance of reconstituted tobacco and the smoking quality of heated cigarettes. Therefore, effective pretreatment of tobacco stem fibers to remove lignin and improve fiber quality is crucial for enhancing the quality of reconstituted tobacco and the utilization rate of tobacco stems.

[0003] Existing tobacco stem fiber processing technologies mainly include the following routes: First, chemical treatment, such as acid / alkali pretreatment or eutectic solvent (DES) treatment, can remove lignin, but there are problems with chemical residues and limited functionality; Second, exogenous reinforcing agent addition method, for example, directly adding purchased nanocellulose or wood pulp fiber to the pulp to improve the strength of the sheet base. This method relies on external raw materials and is decoupled from the tobacco stem pretreatment process; Third, nanocellulose additive method, for example, directly preparing nanocellulose from tobacco stems and then adding it back to the pulp. This is essentially still an exogenous addition mode, separating the preparation and use processes; Fourth, enzymatic pretreatment, for example, using ferulic acid esterase / cellulase to perform biological enzyme pretreatment on tobacco stems and then mechanically nano-sized, but the enzyme treatment function is limited and does not involve the imparting of antibacterial function.

[0004] Microbial fermentation of tobacco is currently a hot research topic. Numerous studies have shown that treating tobacco leaves with appropriate microbial agents can effectively increase aroma and reduce irritation. In recent years, microbial fermentation has been gradually extended to tobacco stems / shreds. Tobacco stems have a high content of cell wall substances, including cellulose, hemicellulose, pectin, and lignin. Microorganisms can produce laccases and cellulases that can degrade lignin, cellulose, and hemicellulose, which is an important way to improve the usability and sensory quality of tobacco stems.

[0005] For example, patent document CN109602070A discloses the application of the SMXP-58 strain in reducing the cellulose content of tobacco stems. This involves preparing a microbial agent containing live bacteria from the Bacillus subtilis SMXP-58 strain, mixing it with tobacco stems, and then fermenting it to reduce the cellulose content. Patent document CN116711876A discloses a method for solid-state fermentation of tobacco stems using Bacillus. This method involves screening for endogenous Bacillus in the tobacco stems for fermentation, and then spraying the fermentation broth onto the tobacco stems for solid-state fermentation to degrade the cellulose. However, these two existing technologies only disclose the application of different Bacillus strains in degrading cellulose in tobacco stems. In the subsequent reconstituted tobacco leaf or cigarette manufacturing process, they cannot simultaneously complete the lignin degradation and softening of fibers, the in-situ generation of antimicrobial peptides, and the direct mechanical nano-sizing treatment after fermentation to generate self-derived cellulose nanofibers / nanocrysts in situ. Existing technologies typically achieve this by adding chemical preservatives or antibacterial agents such as chitosan and nano-silver during the coating stage, which is a "post-treatment" rather than "intrinsic fiber modification." This approach results in antibacterial agents being distributed only on the substrate surface, easily detaching, having poor durability, and increasing the burden on the coating process.

[0006] Therefore, there is an urgent need to provide a method for preparing reconstituted tobacco leaves based on microbial fermentation, which can achieve comprehensive modification of tobacco stem fibers by "one microorganism for two uses and fermentation-nanotechnology integration", so that the fibers treated by microorganisms can be directly used for reconstituted tobacco leaf making, significantly improving the strength and antibacterial properties of the leaf base. Summary of the Invention

[0007] The purpose of this application is to provide a method for preparing reconstituted tobacco leaves based on microbial fermentation, as well as reconstituted tobacco leaves and heated cigarettes, to achieve comprehensive modification of tobacco stem fibers by "one microorganism for two uses and fermentation-nanotechnology integration", so that the fibers treated by microorganisms can be directly used for reconstituted tobacco leaf making, significantly improving the strength and antibacterial properties of the leaf base.

[0008] To solve the above technical problems: This application provides a method for preparing reconstituted tobacco leaves based on microbial fermentation, comprising the following steps: Step A: Fermentation is carried out using microbial strains. After centrifugation, fermentation supernatant and bacterial cells are obtained. The bacterial cells are then resuspended to obtain inoculum. Step B: Ferment the tobacco stem material using the inoculum to obtain the fermentation product; after recycling the fermentation product, a suspension is obtained, which is then heated to inactivate the microorganisms and enzymes in the suspension, thus obtaining the tobacco stem fiber material. Step C: Mix tobacco stem fibers with tobacco dust and pulp them. After papermaking, pressing and drying, dry sheet base is obtained. Concentrate the fermentation supernatant to obtain coating liquid. Coat the dry sheet base with the coating liquid and dry it to obtain reconstituted tobacco leaves.

[0009] Furthermore, the microbial strain includes one or a combination of two of the following: Lactobacillus plantarum L8 strain or engineered Bacillus subtilis strain.

[0010] Furthermore, in step A, the seed culture medium for Lactobacillus plantarum strain L8 is MRS liquid medium, and the fermentation culture conditions are: anaerobic conditions, temperature of 30-45℃, pH of 5.5-7.0, rotation speed of 50-200 rpm, and culture time of 24-72 h.

[0011] Furthermore, the seed culture medium for the engineered Bacillus subtilis strain is LB liquid medium, and the fermentation culture conditions are as follows: under aerobic conditions, solubility is greater than or equal to 30%, temperature is 30-45℃, pH value is 5.5-7.0, rotation speed is 50-200 rpm, and culture time is 24-72 h.

[0012] Furthermore, the concentration of the inoculum solution is greater than or equal to 1×10⁻⁶. 8 CFU / mL.

[0013] Furthermore, in step B, the solid-liquid ratio of the inoculum to the tobacco stem material is 1:(0.1-0.2), 1:(0.2-0.5), 1:(0.5-0.8), 1:(0.8-1.2), 1:(1.2-2.0), or 1:(2.0-3.0), where the unit for solid is grams and the unit for liquid is milliliters.

[0014] Furthermore, the suspension contains tobacco stem matrix fibers, in-situ exfoliated cellulose nanofibers, and nanocrystals.

[0015] Furthermore, the cellulose nanofibers have a diameter of 3-15 nm and a length of 1-8 μm; the nanocrystals are rod-shaped cellulose nanocrystals with a diameter of 5-20 nm and a length of 100-400 nm.

[0016] Furthermore, in step C, the mass ratio of tobacco stem fiber to tobacco dust is (2-4):(6-8); the moisture content of the dried sheet base is 15-20%, and the quantitative content of the dried sheet base is 62±2 g / m³. 2 .

[0017] Furthermore, the coating solution is obtained by concentrating the fermentation supernatant to a solid content of 30-45%, and the coating amount of the coating solution is 30-38% of the mass of the dried substrate.

[0018] Furthermore, the reconstituted tobacco leaves exhibited inhibition rates of over 90% against Staphylococcus aureus, Escherichia coli, and Aspergillus niger.

[0019] Furthermore, the reconstituted tobacco leaves achieved an inhibition rate of 96.0% or higher against Staphylococcus aureus, 94.8% or higher against Escherichia coli, and 92.5% or higher against Aspergillus niger.

[0020] Another aspect of this application provides a reconstituted tobacco leaf, which is prepared by a method for preparing reconstituted tobacco leaf based on microbial fermentation.

[0021] Another aspect of this application provides a heated cigarette, comprising reconstituted tobacco prepared by a method for preparing reconstituted tobacco based on microbial fermentation.

[0022] In some preferred embodiments, the tobacco matrix segment is also referred to as the aerosol-generating matrix segment, and the aerosol-generating matrix in the aerosol-forming matrix segment includes tobacco. For example, the aerosol-forming material can be formed from sheets of homogeneous tobacco.

[0023] Alternatively or additionally, the aerosol forming matrix may include tobacco-free aerosol forming materials.

[0024] For example, the aerosol forming material can be a sheet that includes nicotine salts and an aerosol forming agent.

[0025] Alternatively or additionally, the aerosol forming matrix may include a single aerosol forming agent. Alternatively, the aerosol forming matrix may include a combination of two or more aerosol forming agents.

[0026] Preferably, the aerosol forming matrix has an aerosol forming agent content of more than 5% by dry weight. More preferably, the aerosol forming matrix may have an aerosol forming agent content between about 5% and about 30% by dry weight. In one embodiment, the aerosol forming matrix has an aerosol forming agent content of about 20% by dry weight.

[0027] In this patent, "aerosol forming agent" is used to describe any suitable known compound or mixture of compounds that promotes aerosol formation in use and is substantially resistant to thermal degradation at the operating temperature of the aerosol-generated article.

[0028] Suitable aerosol forming agents are known in the art and include, but are not limited to: polyols, such as propylene glycol, triethylene glycol, 1,3-butanediol, and glycerol; esters of polyols, such as glycerol monoacetate, glycerol diacetate, or glycerol triacetate; and aliphatic esters of mono-, di-, or polycarboxylic acids, such as dimethyl dodecanoate and dimethyl tetradecanoate. Preferred aerosol forming agents are polyols or mixtures thereof, such as propylene glycol, triethylene glycol, 1,3-butanediol, and most preferably glycerol.

[0029] Preferably, the aerosol forming matrix includes a solid aerosol forming matrix, which may include one or more of fragments, strips, bars or sheets, and contains one or more of herbaceous plant leaves, tobacco leaves, tobacco ribs, flat tobacco and homogeneous tobacco.

[0030] Alternatively, the solid aerosol forming matrix can be placed on or embedded in a thermally stable carrier.

[0031] The carrier can be any form, such as fragments, strips, bars, or sheets.

[0032] Alternatively, the solid aerosol forming matrix can be arranged on the surface of the carrier in the form of, for example, sheets, foams, gels or slurries.

[0033] The aerosol forming matrix can be, by choice, in the form of a plug.

[0034] Alternatively, the plug may include an aerosol-forming material defined by paper or other packaging material. In the case where the aerosol-forming matrix is ​​in the form of a plug, the entire plug comprising any packaging paper is considered to be the aerosol-forming matrix.

[0035] Preferably, the heated cigarette includes an aerosol forming matrix, a support element, an aerosol cooling element, and a filter section. The support element and the aerosol cooling element form an intermediate composite section, while the aerosol forming matrix forms the smoke-generating section.

[0036] Preferably, the aerosol cooling element can have a length of approximately 300 mm per millimeter. 2 With a length of approximately 1000 mm per millimeter 2 The total surface area between them. In a preferred embodiment, the aerosol cooling element has approximately 500 mm per millimeter of length. 2 The total surface area.

[0037] Preferably, the aerosol cooling element has low suction resistance. That is, preferably, the aerosol cooling element provides low resistance to air passing through the aerosol-generated article. Preferably, the aerosol cooling element has virtually no impact on the suction resistance of the aerosol-generated article.

[0038] Preferably, the aerosol forming matrix, support element, aerosol cooling element, and filter section are generally cylindrical and have a substantially uniform outer diameter. For example, they have an outer diameter of at least 5 mm. Preferably, they have an outer diameter between about 5 mm and about 12 mm, for example, between about 5 mm and about 10 mm, or between about 6 mm and about 8 mm. In a preferred embodiment, the outer diameter is 7.2 mm + / - 10%.

[0039] Preferably, the aerosol forming matrix can have a length between about 5 mm and about 15 mm, for example, between about 8 mm and about 12 mm. In a preferred embodiment, the aerosol forming matrix has a length of about 12 mm.

[0040] Preferably, the support element may comprise a hollow tubular element. The support element may be formed from any suitable material or combination of materials.

[0041] More preferably, the support element may be formed from one or more materials from the group consisting of: cellulose acetate; paperboard; crimped paper, such as crimped heat-resistant paper or crimped parchment; and polymeric materials, such as low-density polyethylene (LDPE). In a preferred embodiment, the support element comprises a medium cellulose acetate tube.

[0042] Preferably, the filter section can have a length between approximately 5 mm and approximately 20 mm. In a preferred embodiment, the filter section has a length of approximately 14 mm. The filter section can have a length between approximately 5 mm and approximately 14 mm. In a preferred embodiment, the filter section has a length of approximately 7 mm.

[0043] Compared with existing technologies, this patent has the following beneficial effects: This application provides a method for preparing reconstituted tobacco leaves based on microbial fermentation, which integrates reconstituted tobacco leaves and heated cigarettes. It overcomes the shortcomings of existing tobacco stem fiber processing technologies, such as single function, process separation, strong chemical dependence, and low utilization rate of all components. This method provides an integrated treatment method for tobacco stem fibers based on microbial fermentation. The method uses microbial strains capable of simultaneously producing cellulase / hemipolease and antimicrobial peptides to pre-treat tobacco stem fibers through fermentation. During fermentation, lignin degradation softens the fibers, antimicrobial peptides are loaded in situ to impart antimicrobial function, and enzymatic hydrolysis of the non-crystalline cellulose region pre-creates nano-sites. After fermentation, mechanical nano-sizing is directly performed to generate self-derived cellulose nanofibers / nanocrysts in situ, achieving comprehensive modification of tobacco stem fibers through "one microorganism, two uses; fermentation-nano-sizing integration." The treated fibers can be directly used in reconstituted tobacco leaf making, significantly improving the leaf base strength and antimicrobial properties. Attached Figure Description

[0044] The above description of this application and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0045] Figure 1 This is a microscopic observation of the antimicrobial peptide activity of the fermentation supernatant in Example 1 (where the scale bar is 10 mm). Figure 2The image shows an electron microscope image of the pretreated tobacco stem fibers in Example 1 (where the scale bar is 0.5 µm). Figure 3 This is a microscopic observation of the antimicrobial peptide activity of the fermentation supernatant in Example 2 (where the scale bar is 10 mm). Figure 4 The image shows an electron microscope image of the pretreated tobacco stem fibers in Example 2 (where the scale bar is 100 nm). Figure 5 The image shows an electron microscope image of the pretreated tobacco stem fibers in Example 3 (where the scale bar is 100 nm). Detailed Implementation

[0046] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0047] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings: As used in this application, the term "MRS liquid culture medium" refers to a commercially available culture medium prepared according to the method described in publication number CN107619807A. The specific formulation of 1 L (liter) of MRS liquid culture medium is as follows: add 10 g of peptone, 8 g of beef powder, 4 g of yeast powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, 2 g of triammonium citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, and 1 g of Tween 80 to 800 mL of deionized water, then bring the volume to 1 L, sterilize in an autoclave at 115°C for 20 min, and use after cooling.

[0048] As used in this application, the term "LB liquid medium" refers to a commercially available medium. The specific formulation of 1 L (liter) of LB liquid medium is as follows: add 10 g of trypsinized peptone, 5 g of yeast extract, and 10 g of NaCl to 900 mL of deionized water, stirring until the solutes are completely dissolved, then bring the volume to 1 L with deionized water, sterilize in an autoclave at 121°C for 15 min, and use after cooling.

[0049] As used in this application, the term "coating" refers to the process of uniformly applying a prepared coating solution onto a substrate so that the substrate can fully and evenly absorb the coating solution.

[0050] As used in this application, the term "quantitative" refers to the determination of the oven-dry weight per unit area of ​​reconstituted tobacco leaves according to prescribed test methods, expressed in grams per square meter (g / m²).2 )express.

[0051] As used in this application, the term "thickness" refers to the distance between two opposite surfaces of a reconstituted tobacco leaf under conditions specified by a standard test method, expressed in millimeters (mm).

[0052] As used in this application, the term "tensile strength" refers to the maximum tension that a reconstituted tobacco leaf per unit width can withstand before breaking when stretched at a constant speed using a tensile strength tester, expressed in Newtons per meter (kN / m).

[0053] As used in this application, the term "elongation" refers to the percentage of the elongation at break of a reconstituted tobacco leaf of a specified size, measured by stretching it to break at a constant speed using a tensile strength tester, relative to the original length of the reconstituted tobacco leaf, expressed as a percentage.

[0054] As used in this application, the term "folding endurance" refers to the number of folds that reconstituted tobacco leaves can withstand under a certain tension, as determined by a folding endurance meter, and is measured in units of folds.

[0055] As used in this application, the term "filling value" refers to the volume of reconstituted tobacco per unit mass under a certain pressure, specified by a standard test method, expressed in cubic centimeters per gram (cm³). 3 / g) indicates.

[0056] As used in this application, "freezing degree" refers to the degree of beating, expressed as °SR, which reflects the effects of refining processes such as fiber cutting, splitting, swelling, and hydration after the pulp has passed through a refiner.

[0057] As used in this application, the term "inhibition rate against Staphylococcus aureus" refers to the percentage of Staphylococcus aureus inhibition by the reconstituted tobacco prepared in this application under the conditions specified by the standard test method, expressed as a percentage.

[0058] As used in this application, the term "inhibition rate against Escherichia coli" refers to the percentage of Escherichia coli inhibition achieved by the reconstituted tobacco prepared in this application under the conditions specified by the standard test method, expressed as a percentage.

[0059] As used in this application, the term "inhibition rate against Aspergillus niger" refers to the percentage of inhibition of Aspergillus niger by the reconstituted tobacco prepared by this application under the conditions specified by the standard test method, expressed as a percentage.

[0060] All figures used to represent component amounts, properties (e.g., weight-average molecular weight), reaction conditions, etc., should be considered to be modified in all cases by the terms "within the unavoidable margin of error" or "about". Therefore, the numerical values ​​set forth herein are approximate and may vary depending on the desired properties sought to be obtained in this application. The principle of equivalents, which is applied to a minimum and not intended to limit the scope of the claims, should be applied, for example, each value should be interpreted at least according to the reported significant digits and by applying conventional rounding techniques.

[0061] All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, in particular, meaning that one of ordinary skill in the art can directly and without doubt determine how the technical solution of this application can be implemented after reading the claims, description and drawings of this application.

[0062] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, description, and drawings of this application, those skilled in the art can still arrive at the only correct understanding by reading the claims, description, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this application.

[0063] The "range" disclosed herein is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0064] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0065] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0066] Unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0067] Unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0068] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0069] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0070] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0071] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0072] In this document, unless otherwise stated, “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture.

[0073] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0074] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0076] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of this application are conventional methods, and the materials, reagents, instruments, and equipment used in the following embodiments are commercially available unless otherwise specified.

[0077] Source of strain: (1) The L8 strain of Lactobacillus plantarum was prepared according to the patent document with publication number CN107619807A, and can also be obtained from the China Center for Type Culture Collection, with accession number CCTCC M2017457. The L8 strain of Lactobacillus plantarum produces plantarum antimicrobial peptides through metabolism, which have broad-spectrum antibacterial activity; at the same time, it can secrete cellulase and hemicellulase, which can moderately hydrolyze tobacco stem cellulose.

[0078] (2) The engineered Bacillus subtilis strain was purchased from Beijing Bio-Bio Biotechnology Co., Ltd. The engineered Bacillus subtilis strain can simultaneously express lignin-degrading enzyme system (laccase, manganese peroxidase, lignin peroxidase) and antimicrobial peptide synthase gene clusters, and can metabolize to produce subtilisin and surfactant, which have broad-spectrum antibacterial and antifungal activities.

[0079] This application provides a method for preparing reconstituted tobacco leaves based on microbial fermentation, the reconstituted tobacco leaves, and heated cigarettes, including the following steps: S1. High-density fermentation of microbial strains, the specific steps of which are as follows: (1) Inoculate the microbial strain into the seed culture medium at a volume ratio of 1:1000 and place it in a fully automated bioreactor for high-density fermentation. Set the temperature of the fully automated bioreactor to 30-45℃, the pH value to 5.5-7.0, and the dissolved oxygen level to be adjusted according to the microbial strain. Set the rotation speed to 50-200 rpm and the culture time to 24-72 h to obtain the fermentation broth. The concentration of the fermentation broth is ≥1×10⁻⁶. 9CFU / mL.

[0080] The microbial strains were either Lactobacillus plantarum L8 or engineered Bacillus subtilis. When the microbial strain was Lactobacillus plantarum L8, MRS liquid medium (commercially available) was used as the seed medium for high-density fermentation culture, and the dissolved oxygen was set to an anaerobic state. When the microbial strain was engineered Bacillus subtilis, LB liquid medium (commercially available) was used as the seed medium for high-density fermentation culture, and the dissolved oxygen was set to an aerobic state with DO ≥ 30%.

[0081] (2) The fermentation broth obtained from high-density fermentation in step (1) above is centrifuged in a refrigerated centrifuge at 4℃ and 8000r / min for 5-15 min to obtain the fermentation supernatant and bacterial cells. Here, the bacterial cells are collected and resuspended in sterile physiological saline to achieve an inoculum concentration of 1×10⁻⁶. 8 -1×10 9 CFU / mL was used to obtain the bacterial inoculum.

[0082] S2. Tobacco stem fiber processing, the specific steps are as follows: (1) After removing impurities and washing the tobacco stem raw material, it is placed in a vibrating fluidized bed dryer for hot air drying to obtain the dried tobacco stem raw material.

[0083] The hot air drying temperature is 58-60℃, 60-64℃, 64-68℃ or 68-72℃, and the moisture content of the dried tobacco stem raw material is ≤5%. This can be understood as the moisture content of the dried tobacco stem raw material being 5%, or 4.5%, 4.0% or 3.0%, etc.

[0084] (2) The dried tobacco stem raw material obtained in step S2 (1) above is placed in a hammer mill for crushing. The screen aperture of the hammer mill is adjusted to 2.0-10.0 mm. After passing through the screen, it is sent into the silo and sterilized for 15 min under steam conditions at 121°C using a continuous spiral sterilizer. After cooling to room temperature, it is ready for use. The sterilized tobacco stem material is obtained to provide raw materials for the subsequent solid-state fermentation of tobacco stems.

[0085] In some other specific embodiments, the dried tobacco stem raw material obtained in step S2 (1) above can also be placed in a hammer mill for crushing. The screen aperture of the hammer mill is adjusted to ≤1.0 mm. After passing through the screen, it is sent into the silo and sterilized for 15 min under steam conditions at 121°C using a continuous spiral sterilizer. After cooling to room temperature, it is ready for use to obtain sterilized tobacco stem material, which provides raw material for subsequent liquid fermentation of tobacco stem.

[0086] (3) The sterilized tobacco stem material obtained in step (2) of step S2 above is mixed with the inoculum obtained in step S1 above at a solid-liquid ratio of 1:(0.1-3.0), and transferred to a fully automatic solid-state fermenter or a stirred bioreactor. The fermentation temperature is set to 30-45℃, the pH value is set to 5.5-7.0 (adjusted by automatic acid / alkali addition), the aeration rate (aerobic stage) is set to maintain DO≥30% for aerobic bacteria, the rotation speed is 50-200 rpm, the exhaust gas O2 / CO2, redox potential and cellulase activity are monitored online during the fermentation process, the fermentation time is 24-120 h (set according to the strain and stage), and the antimicrobial peptide content is detected by sampling every 6 hours through an automatic sampling system (using high performance liquid chromatography-mass spectrometry), and the fermentation product is finally obtained.

[0087] It can be understood that the solid-liquid ratio of the sterilized tobacco stem material obtained in step S2 (2) to the inoculum obtained in step S1 is 1:(0.1-3.0), which can include any ratio range of 1:(0.1-0.2), 1:(0.2-0.5), 1:(0.5-0.8), 1:(0.8-1.2), 1:(1.2-2.0), or 1:(2.0-3.0). The unit of solid is grams, and the unit of liquid is milliliters.

[0088] (4) The fermentation product obtained in step (3) of S2 above does not need to be inactivated and is directly transferred to a high-pressure microfluidic nano-homogenizer for circulation processing. This equipment can generate a shear force of up to 200 MPa, and the processing chamber is a diamond-interactive cavity. The working pressure of the high-pressure microfluidic nano-homogenizer is set to 80-150 MPa, the number of circulation processes is 3-8, and the slurry temperature is controlled within the range of ≤30℃ by the built-in cooling jacket. After circulation processing, a milky white suspension is obtained.

[0089] By sampling the suspension, the particle size and distribution of nanocellulose were characterized using a laser particle size-Zeta potential analyzer, and the morphology of the nanofibers was observed using a transmission electron microscope. The results showed that the suspension contained tobacco stem matrix fibers and in-situ exfoliated cellulose nanofibers (CNF) / nanocrysts (CNC). Cellulose nanofibers with diameters of 3-15 nm and lengths of 1-8 μm and rod-shaped cellulose nanocrystals with diameters of 5-20 nm and lengths of 100-400 nm were visible.

[0090] (5) Heat the suspension obtained in step S2 (4) above in a water bath at 85-90℃ for 25-30 min to deactivate microorganisms and residual enzymes, and obtain tobacco stem fiber pulp.

[0091] Further testing revealed that the antimicrobial activity retention rate of the antimicrobial peptides after heat treatment was ≥80%.

[0092] S3. Preparation of reconstituted tobacco leaves, the specific steps are as follows: (1) The tobacco stem fiber pulp prepared in step S2 (5) above is mixed with tobacco dust (crushed to ≤0.5 mm) in a mixing tank at a mass ratio of (2-4):(6-8). Deionized water is added to adjust the concentration of the mixed pulp to 2-3%. Then, 8-15% of external fibers are added. The pulp is then decomposed using a high-consistency hydraulic pulper. The rotation speed of the high-consistency hydraulic pulper is adjusted to 1000-1200 r / min, 1200-1300 r / min, or 1300-1500 r / min, and the decomposition time is 10-12 min, 12-13 min, 13-15 min, or 15-20 min. After decomposition, the pulp is pumped into a double-disc refiner for beating. The disc gap of the double-disc refiner is adjusted to 0.1-0.5 mm, and the beating degree is controlled to 28-35°SR (detected online by a Schubert beater) to obtain the pulp.

[0093] (2) The pulp obtained in step S3 (1) above is diluted to a concentration of 0.2-0.5% by a pulp pump and then sent to the inclined wire forming device of the papermaking reconstituted tobacco production line, so that the second pulp is dehydrated under the action of gravity and vacuum suction (-0.02 to -0.05MPa) to form a continuous wet paper web.

[0094] The tilt angle of the inclined wire forming device is adjusted to 15-30°, the mesh size is 80-100 mesh, the running speed of the forming wire is 10-30 m / min, and the wet paper web dryness is about 18-22%.

[0095] (3) The continuous wet paper web obtained in step (2) of step S3 above is fed into a three-roll two-pressure zone press through a felt. The pressure of the first pressing line is set to 0.3-0.5 MPa and the pressure of the second pressing line is set to 0.5-0.8 MPa. After pressing, the dryness is increased to 40-45%, and the sheet base is obtained.

[0096] (4) The film substrate obtained in step (3) of S3 above is sent into a multi-stage hot air penetration drying oven. The drying temperature is set to 90-120℃, the hot air velocity is 5-15 m / s, and the film substrate is dried until the moisture content is 15-20%. The quantitative control of the film substrate is 62±2 g / m³. 2 (Monitored by an online quantitative analyzer), thus obtaining the dried film base.

[0097] (5) The fermentation supernatant obtained in step (2) of S1 above is concentrated to a solid content of 30-45% using a vacuum rotary evaporator. The concentrated coating liquid is uniformly coated onto the surface of the substrate using a precision coating machine with a metering rod coating method, and the coating amount is 30-38% of the dry substrate mass. The coated substrate is sent to a suspended hot air drying oven, and the drying temperature is set to 100-110℃, the wind speed is 10-20 m / s, and the moisture content is dried to 10-12% to obtain reconstituted tobacco leaves.

[0098] S4. Preparation of heated cigarettes, the specific steps are as follows: The reconstituted tobacco leaves obtained in step S3 (5) above are cut and sent to a constant temperature and humidity chamber (22±1℃, 60±3%RH) for equilibration for 48 h. After being shredded, they are used as tobacco matrix. The tobacco matrix is ​​rolled with cigarette paper to obtain tobacco matrix segments, which are then connected with support segments and filter segments to obtain heated cigarette sticks.

[0099] Example 1

[0100] (1) Lactobacillus plantarum strain L8 was inoculated into MRS liquid medium at a volume ratio of 1:1000 and placed in a fully automated bioreactor for high-density fermentation. The temperature of the fully automated bioreactor was set at 37℃, the pH value was set at 5.5-7.0, the rotation speed was set at 200 rpm, and the culture was carried out under anaerobic conditions for 24 h until the logarithmic growth phase was reached to obtain the fermentation broth. The concentration of the fermentation broth was ≥1×10⁻⁶. 8 CFU / mL.

[0101] (2) The fermentation broth obtained in step (1) above was centrifuged for 7 min at 4℃ and 8000 r / min using a refrigerated centrifuge to obtain the fermentation supernatant and bacterial cells. The bacterial cells were collected and resuspended in sterile physiological saline to achieve an inoculum concentration of 1×10⁻⁶. 9 CFU / mL was used to obtain the bacterial inoculum.

[0102] (3) Weigh 500 g of tobacco stem raw material and wash it with water. Place it in a vibrating fluidized bed dryer and dry it with hot air at 60℃. The moisture content of the dried tobacco stem raw material is ≤5%. Crush it to 2.0-5.0 mm with a hammer mill. After sieving, use a continuous spiral sterilizer to sterilize it under steam conditions at 121℃ for 15 min. Cool it to room temperature and use it for later use to obtain sterilized tobacco stem material.

[0103] (4) The sterilized tobacco stem material obtained in step (3) above and the inoculum obtained in step (2) above are mixed in a fermentation pan at a solid-liquid ratio of 1:0.1. Sterile water is added to make the moisture content of the tobacco stem reach 55%. After stirring evenly, the initial pH value is adjusted to 6.2 using 1 mol / L NaOH. The fermentation pan is placed in a constant temperature incubator and solid-state fermented at 35℃ for 60 h. During the fermentation, the mixture is turned over once every 12 h to ensure the uniformity and aeration of the material. During the fermentation process, the pH value (dropped to 4.8-5.2) and the antimicrobial peptide activity (inhibition zone detected by agar diffusion method) are sampled regularly to obtain the fermentation product.

[0104] The results are as follows Figure 1 As shown, the antimicrobial peptide activity of fermentation supernatant was detected by agar diffusion method, with Escherichia coli as the indicator bacterium. The fermentation supernatant formed a well-defined inhibition zone with a diameter of 16 mm.

[0105] (5) The fermentation product obtained in step (4) above does not need to be inactivated. It is directly transferred to a high-pressure microfluidic nano homogenizer for circulation treatment. The working pressure of the high-pressure microfluidic nano homogenizer is set to 80 MPa, the number of circulation treatments is 5, the slurry temperature at the outlet is ≤30℃, and a milky white suspension is obtained after circulation treatment.

[0106] By sampling the suspension, the particle size and distribution of nanocellulose were characterized using a laser particle size-Zeta potential analyzer, and the morphology of the nanofibers was observed using a transmission electron microscope.

[0107] The results are as follows Figure 2 As shown, the suspension contains tobacco stem matrix fibers and in-situ exfoliated cellulose nanofibers (CNF) / nanocrysts (CNC), with visible cellulose nanofibers of 5-15 nm in diameter and 1-5 μm in length, as well as rod-shaped cellulose nanocrystals of 10-20 nm in diameter and 100-300 nm in length.

[0108] (6) The suspension obtained in step (5) above is heated in a water bath at 90°C for 25 min to deactivate microorganisms and residual enzymes, and tobacco stem fiber pulp is obtained.

[0109] Further testing revealed that the antimicrobial activity retention rate of the antimicrobial peptides after heat treatment was >85%, indicating that the Lactobacillus plantarum L8 strain has good thermal stability.

[0110] (7) The tobacco stem fiber pulp obtained in step (6) above is mixed with tobacco dust (crushed to ≤0.5 mm) in a mixing tank at a mass ratio of 3:7. Deionized water is added to adjust the concentration of the mixed pulp to 2.5%. Then, 10% wood pulp fiber (external fiber) is added. The pulp is then decomposed using a high-consistency hydraulic pulper. After decomposition, the pulp is pumped into a double-disc refiner for pulping treatment. The beating degree is controlled at 30±1°SR to obtain the pulp.

[0111] (8) The pulp obtained in step (7) above is diluted to a concentration of 0.3% by a pulp pump and sent to the inclined wire forming device of the papermaking reconstituted tobacco production line. The inclination angle of the inclined wire forming device is adjusted to 20° and the mesh size is 90 mesh, so that the second pulp is dewatered under the action of gravity and vacuum suction (-0.03 MPa). The running speed of the forming wire is 20 m / min, and the wet paper web dryness is about 20%, forming a continuous wet paper web.

[0112] (9) The continuous wet paper web obtained in step (8) is fed into a three-roll, two-press press. The pressure of the first press line is set to 0.4 MPa, and the pressure of the second press line is set to 0.6 MPa. After pressing, the dryness is increased to 42%, and a sheet base is obtained. The sheet base is sent into a multi-stage hot air penetration drying oven. The drying temperature is set to 105℃, and the hot air velocity is set to 10 m / s. The sheet base is dried until the moisture content is 18%. The basis weight of the sheet base is controlled to be 62±2 g / m³. 2 This yields the dried film base.

[0113] (10) The fermentation supernatant obtained in step (2) above is concentrated to a solid content of 40% using a vacuum rotary evaporator. The concentrated coating liquid is uniformly coated onto the surface of the substrate using a precision coating machine with a metering rod coating method. The coating amount is 35% of the dry substrate mass. The coated substrate is sent to a suspended hot air drying oven, and the drying temperature is set to 105℃, the drying time is 6 min, the wind speed is 15 m / s, and the moisture content is 11% to obtain reconstituted tobacco leaves.

[0114] (11) After the reconstituted tobacco leaves obtained in step (10) are cut, they are sent to a constant temperature and humidity room (22±1℃, 60±3%RH) for equilibration for 48 h. After being shredded, they are used as tobacco matrix. The tobacco matrix is ​​rolled with cigarette paper to obtain tobacco matrix segments, which are then connected with support segments and filter segments to obtain heated cigarette sticks.

[0115] Example 2

[0116] The difference from Example 1 is that this example uses an engineered strain of Bacillus subtilis for fermentation.

[0117] (1) The engineered Bacillus subtilis bacteria were inoculated into LB liquid medium at a volume ratio of 1:1000 and cultured in a shaker at 37°C and 200 rpm for 24 h until the logarithmic growth phase was reached, yielding a fermentation broth with a concentration ≥1×10⁻⁶. 8 CFU / mL.

[0118] (2) The fermentation broth obtained in step (1) above was centrifuged for 7 min at 4℃ and 8000 r / min using a refrigerated centrifuge to obtain the fermentation supernatant and bacterial cells. The bacterial cells were collected and resuspended in sterile physiological saline to achieve an inoculum concentration of 1×10⁻⁶. 9 CFU / mL was used to obtain the bacterial inoculum.

[0119] (3) Weigh 500 g of tobacco stem raw material and wash it with water. Place it in a vibrating fluidized bed dryer and dry it with hot air at 60℃. The moisture content of the dried tobacco stem raw material is ≤5%. It is crushed to ≤1 mm by a hammer mill and then screened. It is sterilized for 15 min under steam conditions at 121℃ by a continuous spiral sterilizer. After cooling to room temperature, it is ready for use to obtain sterilized tobacco stem material.

[0120] (4) The sterilized tobacco stem material obtained in step (3) above and the inoculum obtained in step (2) above are mixed in a fermentation pan at a solid-liquid ratio of 1:2.5, so that the inoculum amount reaches ≥1×10 8 CFU / g tobacco stems were placed in a fully automated bioreactor for high-density fermentation. The temperature of the fully automated bioreactor was set at 37℃, the initial pH was 6.8, the aeration rate was 1.0 vvm, the rotation speed was 200 rpm, and the dissolved oxygen was controlled at 30%-40%. Fermentation was carried out under aerobic conditions for 72 h to obtain the fermentation product. The pH value and antimicrobial peptide activity were sampled and monitored regularly (the inhibition zone was detected by the agar diffusion method).

[0121] The results are as follows Figure 3 As shown, the antimicrobial peptide activity of the fermentation supernatant was detected by the agar diffusion method, with Escherichia coli as the indicator bacterium. The fermentation supernatant formed a well-defined inhibition zone with a diameter of 19 mm.

[0122] (5) The fermentation product obtained in step (4) above does not need to be inactivated. It is directly transferred to a high-pressure microfluidic nano homogenizer for circulation treatment. The working pressure of the high-pressure microfluidic nano homogenizer is set to 100 MPa, the number of circulation treatments is 4, the slurry outlet temperature is 25-30℃, and a uniform suspension is obtained after circulation treatment.

[0123] By sampling the suspension, the particle size and distribution of nanocellulose were characterized using a laser particle size-Zeta potential analyzer, and the morphology of the nanofibers was observed using a transmission electron microscope.

[0124] The results are as follows Figure 4 As shown, the suspension contains tobacco stem matrix fibers and in-situ exfoliated cellulose nanofibers (CNF) / nanocrysts (CNC), with visible cellulose nanofibers of 3-10 nm in diameter and 2-8 μm in length, as well as rod-shaped cellulose nanocrysts of 5-15 nm in diameter and 150-400 nm in length.

[0125] (6) The suspension obtained in step (5) above is heated in a water bath at 85°C for 30 min to deactivate microorganisms and residual enzymes, and tobacco stem fiber pulp is obtained.

[0126] Further testing revealed that the antimicrobial activity retention rate of the antimicrobial peptides after heat treatment was approximately 80%, indicating that the Bacillus subtilis engineered bacteria inoculation exhibited good thermal stability.

[0127] (7) The tobacco stem fiber pulp obtained in step (6) above is mixed with tobacco dust (crushed to ≤0.5 mm) in a mixing tank at a mass ratio of 3:7. Deionized water is added to adjust the concentration of the mixed pulp to 2.5%. Then, 10% wood pulp fiber (external fiber) is added. The pulp is then decomposed using a high-consistency hydraulic pulper. After decomposition, the pulp is pumped into a double-disc refiner for pulping treatment. The beating degree is controlled at 30±1°SR to obtain the pulp.

[0128] (8) The pulp obtained in step (7) above is diluted to a concentration of 0.3% by a pulp pump and sent to the inclined wire forming device of the papermaking reconstituted tobacco production line. The inclination angle of the inclined wire forming device is adjusted to 20° and the mesh size is 90 mesh, so that the second pulp is dewatered under the action of gravity and vacuum suction (-0.03 MPa). The running speed of the forming wire is 20 m / min, and the wet paper web dryness is about 20%, forming a continuous wet paper web.

[0129] (9) The continuous wet paper web obtained in step (8) is fed into a three-roll, two-press press. The pressure of the first press line is set to 0.4 MPa, and the pressure of the second press line is set to 0.6 MPa. After pressing, the dryness is increased to 42%, and a sheet base is obtained. The sheet base is sent into a multi-stage hot air penetration drying oven. The drying temperature is set to 105℃, and the hot air velocity is set to 10 m / s. The sheet base is dried until the moisture content is 18%. The basis weight of the sheet base is controlled to be 62±2 g / m³. 2 This yields the dried film base.

[0130] (10) The fermentation supernatant obtained in step (2) above is concentrated to a solid content of 40% using a vacuum rotary evaporator. The concentrated coating liquid is uniformly coated onto the surface of the substrate using a precision coating machine with a metering rod coating method. The coating amount is 35% of the dry substrate mass. The coated substrate is sent to a suspended hot air drying oven, and the drying temperature is set to 105℃, the drying time is 6 min, the wind speed is 15 m / s, and the moisture content is 11% to obtain reconstituted tobacco leaves.

[0131] (11) After the reconstituted tobacco leaves obtained in step (10) are cut, they are sent to a constant temperature and humidity room (22±1℃, 60±3%RH) for equilibration for 48 h. After being shredded, they are used as tobacco matrix. The tobacco matrix is ​​rolled with cigarette paper to obtain tobacco matrix segments, which are then connected with support segments and filter segments to obtain heated cigarette sticks.

[0132] Example 3

[0133] The difference from Example 1 is that this example uses Lactobacillus plantarum strain L8 and Bacillus subtilis engineered strain for fermentation.

[0134] (1) Lactobacillus plantarum strain L8 was inoculated into MRS liquid medium at a volume ratio of 1:1000 and placed in a fully automated bioreactor for high-density fermentation. The temperature of the fully automated bioreactor was set at 37℃, the pH value was set at 5.5-7.0, the rotation speed was set at 200 rpm, and the culture was carried out under anaerobic conditions for 24 h until the logarithmic growth phase was reached to obtain the fermentation broth. The concentration of the fermentation broth was ≥1×10⁻⁶. 8 CFU / mL.

[0135] (2) The fermentation broth obtained in step (1) above was centrifuged for 7 min at 4℃ and 8000 r / min using a refrigerated centrifuge to obtain the fermentation supernatant and bacterial cells. The bacterial cells were collected and resuspended in sterile physiological saline to achieve an inoculum concentration of 1×10⁻⁶. 9 CFU / mL was used to obtain the inoculum of Lactobacillus plantarum strain L8.

[0136] (3) Inoculate the engineered Bacillus subtilis bacteria into LB liquid medium at a volume ratio of 1:1000 and place it in a shaker for incubation. Set the shaker temperature to 37℃ and the rotation speed to 200 rpm, and incubate for 24 h until the logarithmic growth phase to obtain the fermentation broth. The concentration of the fermentation broth is ≥1×10⁻⁶. 8 CFU / mL.

[0137] (4) The fermentation broth obtained in step (3) above is centrifuged for 7 min at 4℃ and 8000 r / min using a refrigerated centrifuge to obtain the fermentation supernatant and bacterial cells. The bacterial cells are collected and resuspended in sterile physiological saline to achieve an inoculum concentration of 1×10⁻⁶. 9 CFU / mL was used to obtain the engineered Bacillus subtilis inoculum solution.

[0138] (5) Weigh 500 g of tobacco stem raw material and wash it with water. Place it in a vibrating fluidized bed dryer and dry it with hot air at 60℃. The moisture content of the dried tobacco stem raw material is ≤5%. It is crushed to ≤1 mm by a hammer mill and then screened. It is sterilized for 15 min under steam conditions at 121℃ by a continuous spiral sterilizer. After cooling to room temperature, it is ready for use to obtain sterilized tobacco stem material.

[0139] (6) The sterilized tobacco stem material obtained in step (5) and the Bacillus subtilis engineered bacteria inoculation solution obtained in step (4) are mixed in a fermentation pan at a solid-liquid ratio of 1:2.0, so that the inoculation amount of bacteria reaches ≥1×10 8 CFU / g tobacco stems were placed in a fully automated bioreactor for high-density fermentation. The temperature of the fully automated bioreactor was set at 37℃, the initial pH value was 6.8, the aeration rate was 1.0 vvm, the rotation speed was 180 rpm, the dissolved oxygen was >30%, and the fermentation was carried out under aerobic conditions for 36 h to obtain fermentation products containing engineered Bacillus subtilis bacteria.

[0140] (7) The fermentation product obtained in step (6) above does not need to be inactivated (not sterilized), and is directly inoculated into the fully automated bioreactor with Lactobacillus plantarum strain L8 (1×10⁻⁶). 7 The aeration was stopped, and the fermentation conditions were changed to anaerobic fermentation. The temperature was set at 35℃, and the pH was naturally reduced (the pH of the fermentation broth gradually decreased from 6.5 to 4.8). The stirring speed was 50 rpm, and the fermentation time was 36 h. The total fermentation time for the two stages was 72 h. The fermentation product containing both Bacillus subtilis engineered bacteria and Lactobacillus plantarum L8 strain was obtained, which was labeled as the total fermentation product.

[0141] (8) The total fermentation product obtained in step (7) above is directly transferred to a high-pressure microfluidic nano homogenizer for circulation treatment. The working pressure of the high-pressure microfluidic nano homogenizer is set to 100 MPa, the number of circulation treatments is 5, the slurry outlet temperature is <30℃, and a uniform suspension is obtained after circulation treatment.

[0142] By sampling the suspension, the particle size and distribution of nanocellulose were characterized using a laser particle size-Zeta potential analyzer, and the morphology of the nanofibers was observed using a transmission electron microscope.

[0143] The results are as follows Figure 5 As shown, the yield of cellulose nanofibers (CNF) / nanocrystals (CNC) exfoliated in situ from the suspension was significantly higher than that of single-strain fermentation in Examples 1-2, and the degree of fiber nanostructuring was also higher.

[0144] (9) The suspension obtained in step (8) above is heated in a water bath at 90°C for 25 min to deactivate microorganisms and residual enzymes, and tobacco stem fiber pulp is obtained.

[0145] Further testing revealed that the antimicrobial activity retention rate of the antimicrobial peptides after heat treatment was approximately 80%, indicating that the Bacillus subtilis engineered bacteria inoculation exhibited good thermal stability.

[0146] (10) The tobacco stem fiber pulp obtained in step (9) above is mixed with tobacco dust (crushed to ≤0.5 mm) in a mixing tank at a mass ratio of 3:7. Deionized water is added to adjust the concentration of the mixed pulp to 2.5%. Then, 10% wood pulp fiber (external fiber) is added. The pulp is then decomposed using a high-consistency hydraulic pulper. After decomposition, the pulp is pumped into a double-disc refiner for pulping treatment. The beating degree is controlled at 30±1°SR to obtain the pulp.

[0147] (11) The pulp obtained in step (10) above is diluted to a concentration of 0.3% by a pulp pump and then sent to the inclined wire forming device of the papermaking reconstituted tobacco production line. The inclination angle of the inclined wire forming device is adjusted to 20° and the mesh size is 90 mesh, so that the second pulp is dewatered under the action of gravity and vacuum suction (-0.03 MPa). The running speed of the forming wire is 20 m / min, and the wet paper web dryness is about 20%, forming a continuous wet paper web.

[0148] (12) The continuous wet paper web obtained in step (11) is fed into a three-roll, two-press press. The pressure of the first press line is set to 0.4 MPa, and the pressure of the second press line is set to 0.6 MPa. After pressing, the dryness is increased to 42%, and a sheet base is obtained. The sheet base is sent into a multi-stage hot air penetration drying oven. The drying temperature is set to 105℃, and the hot air velocity is set to 10 m / s. The sheet base is dried until the moisture content is 18%. The basis weight of the sheet base is controlled to be 62±2 g / m³. 2 This yields the dried film base.

[0149] (13) The fermentation supernatant obtained in step (2) above is concentrated to a solid content of 40% by a vacuum rotary evaporator. The concentrated coating liquid is uniformly coated onto the surface of the substrate using a precision coating machine with a metering rod coating method. The coating amount is 35% of the dry substrate mass. The coated substrate is sent to a suspended hot air drying oven, and the drying temperature is set to 105℃, the drying time is 6 min, the wind speed is 15 m / s, and the moisture content is 11% to obtain reconstituted tobacco leaves.

[0150] (14) After the reconstituted tobacco leaves obtained in step (13) are cut, they are sent to a constant temperature and humidity room (22±1℃, 60±3%RH) for equilibration for 48 h. After being shredded, they are used as tobacco matrix. The tobacco matrix is ​​rolled with cigarette paper to obtain tobacco matrix segments, which are then connected with support segments and filter segments to obtain heated cigarette sticks.

[0151] Comparative Example 1

[0152] Compared with Example 1, the difference is that this comparative example uses the hot water extraction process in the papermaking reconstituted tobacco leaf process of the prior art, without any microbial fermentation or nano-processing. Specifically, 500 g of tobacco stems are taken, washed, dried, and crushed into 2-5 mm pieces. Hot water at 60°C is added at a solid-liquid ratio of 1:4, and the mixture is soaked and extracted for 45 minutes. The residue is then filtered to obtain tobacco stem residue. After preliminary defiberization, the residue is mixed with tobacco dust at a ratio of tobacco stem:to tobacco dust = 3:7, and 10% external fiber (wood pulp fiber) is added. The mixture is diluted with an appropriate amount of water to a pulp concentration of 2.5%, and then beaten in a trough beater to a freeness of 30±1°SR to obtain tobacco stem fiber pulp. This pulp is then sheeted, coated, and dried to prepare reconstituted tobacco leaves. The reconstituted tobacco leaves are then shredded and rolled to obtain heated cigarettes.

[0153] Comparative Example 2

[0154] Compared with Comparative Example 1, the difference is that this comparative example uses the method described in the patent document with publication number CN107217532A, using commercially available wood pulp as raw material, hydrolyzing it with 64 wt% sulfuric acid at 45°C for 30 min, then terminating the reaction by washing with a large amount of water, centrifuging and dialysis to neutrality, and ultrasonically dispersing to obtain an exogenous wood pulp nanocellulose crystal (CNC) suspension with a concentration of approximately 2%. After preliminary defibrination of tobacco stem residue, it is mixed with tobacco dust at a ratio of tobacco stem:to tobacco dust = 3:7, with 10% exogenous fiber (wood pulp fiber) added, and diluted with an appropriate amount of water to a pulp concentration of 2.5%. Simultaneously, the above-mentioned exogenous wood pulp nanocellulose crystal (CNC) suspension is added to the pulping tank, so that the amount of nanocellulose added is 5% of the dry weight of the tobacco stem. The pulp is then beaten in a trough beater to a freeness of 30±1°SR to obtain tobacco stem fiber pulp. This pulp is then sheeted, coated, and dried to prepare reconstituted tobacco leaves. The reconstituted tobacco leaves are then shredded and rolled to obtain heated cigarettes.

[0155] Comparative Example 3

[0156] Compared with Example 3, the difference is that this comparative example uses the method described in the patent document with publication number CN107619807A, which only uses Lactobacillus plantarum strain L8 for fermentation, and then prepares reconstituted tobacco leaves through subsequent sheeting, coating and drying. The reconstituted tobacco leaves are then cut into shreds and rolled into heated cigarettes.

[0157] Comparative Example 4

[0158] Compared with Example 3, the difference is that this comparative example uses the method described in the patent document with publication number CN116711876A, which only uses Bacillus for fermentation, and then prepares reconstituted tobacco leaves through subsequent sheeting, coating and drying. The reconstituted tobacco leaves are then cut into shreds and rolled into heated cigarettes.

[0159] Test case

[0160] To further illustrate the advantages of this embodiment, the reconstituted tobacco leaves prepared in Examples 1-3 and Comparative Examples 1-3 were further tested according to GB / T 22898-2008 for tensile strength and elongation; according to GB / T 457-2008 for folding endurance; according to YC / T 16.3-2003 for filling value; and according to GB / T 20944.3-2008 for inhibition rate against Staphylococcus aureus, Escherichia coli and Aspergillus niger by shaking method.

[0161] The specific results are shown in Table 1. Compared with the reconstituted tobacco leaves of the comparative examples, the embodiments of this application have achieved significant improvements in mechanical properties, antibacterial durability, process economy, and environmental friendliness. Since comparative examples 3 and 4 have similar results to comparative example 2, with significant improvements of 40-45% in the inhibition rates against Staphylococcus aureus, Escherichia coli, and Aspergillus niger, they are not shown in Table 1. Only comparative example 2 is used to describe the results.

[0162] In the embodiments, the tensile strength of heated cigarettes can reach 1.5-1.8 kN / m, which is more than 200% higher than that of untreated tobacco stems (about 0.4-0.5 kN / m) and more than 50% higher than that of the exogenous nanocellulose addition route (about 1.0-1.1 kN / m) or single strain fermentation. The amount of wood pulp fiber added is reduced from the conventional 10-30% to 0, achieving complete self-sufficiency, significantly reducing raw material costs and eliminating wood odor. The in-situ loaded antimicrobial peptides endow the fiber with intrinsic antimicrobial properties, with an inhibition rate of ≥95% against Staphylococcus aureus, Escherichia coli and Aspergillus niger, effectively solving the problem of mold growth during storage. No strong acids, strong alkalis or chemical antimicrobial agents are used throughout the process. The fermentation supernatant can be concentrated and reused in the coating liquid. The overall processing cost is reduced by 30-50% compared with the existing routes. Furthermore, the delignification softening, antimicrobial endowment and nanocellulose preparation are integrated into one process, which is highly integrated and has significant advantages for industrial promotion.

[0163] In Example 3, the combined fermentation of two different microbial strains yielded the most significant results. In contrast, the single microbial strains in Comparative Examples 3 and 4 failed to achieve inhibition rates of over 90% against Staphylococcus aureus, Escherichia coli, and Aspergillus niger. Furthermore, Lactobacillus plantarum L8 is an anaerobic bacterium, while Bacillus subtilis is an aerobic bacterium. The fermentation processes required for the two strains are different, and those skilled in the art have no motivation to use two different strains for combined fermentation, nor can they predict whether beneficial technical effects will be produced.

[0164] Table 1. Performance Comparison of Reconstituted Tobacco Leaves in Different Examples and Comparative Examples

[0165] Therefore, it can be concluded that the method for preparing reconstituted tobacco leaves based on microbial fermentation, the reconstituted tobacco leaves, and the heated cigarettes provided in this application have the following advantages over the prior art: This application constructs an integrated "fermentation-nanotechnology" self-reinforcement system, employing a bifunctional microbial strain that simultaneously produces cellulase and antimicrobial peptides to ferment tobacco stem fibers. During fermentation, the cellulase moderately hydrolyzes the non-crystalline regions of cellulose, pre-creating exfoliation sites for subsequent mechanical nanotechnology treatment. Simultaneously, the antimicrobial peptides are in situ loaded onto the fiber surface, imparting intrinsic antimicrobial properties. After fermentation, without inactivation, washing, or pH adjustment, the entire fermentation broth is directly homogenized under gradient high pressure. Under the synergistic effect of residual enzymes, organic acids, and mechanical shear force, some cellulose is in situ converted into tobacco stem self-derived cellulose nanoparticles. The fiber / nanocrystalline process integrates lignin removal, fiber softening, antibacterial properties, and in-situ generation of nanocellulose in the same process. The self-derived nanocellulose is homologous to the main fiber and can completely replace exogenous wood pulp fiber. It upgrades the traditional separation mode of "softening first, then strengthening, then antibacterial" to a synergistic processing mode of "one bacteria for two uses, fermentation-nanoification integration". The self-derived nanocellulose is homologous to the main tobacco stem fiber and has better compatibility than exogenous addition. It can completely replace or significantly reduce exogenous wood pulp fiber. At the same time, no strong acids, strong alkalis or chemical antibacterial agents are used throughout the process, making it green and safe.

[0166] The terminology and expressions used herein are for descriptive purposes only and this patent should not be limited to these terms and expressions. The use of these terms and expressions does not imply the exclusion of any illustrative and descriptive equivalent features (or parts thereof), and it should be recognized that various possible modifications should also be included within the scope of the claims. Other modifications, variations, and substitutions may also exist. Accordingly, the claims should be considered to cover all such equivalents.

[0167] Similarly, it should be noted that although this patent has been described with reference to the specific embodiments described above, those skilled in the art should recognize that the above embodiments are only used to illustrate this patent, and various equivalent changes or substitutions can be made without departing from the spirit of this patent. Therefore, any changes or modifications to the above embodiments within the essential spirit of this patent will fall within the scope of the claims of this patent.

Claims

1. A method for preparing reconstituted tobacco leaves based on microbial fermentation, characterized in that, Includes the following steps: Step A: Fermentation is carried out using microbial strains. After centrifugation, fermentation supernatant and bacterial cells are obtained. The bacterial cells are then resuspended to obtain inoculum solution. Step B: Ferment the tobacco stem material using the inoculum to obtain fermentation products; after recycling the fermentation products, a suspension is obtained, which is then heated to inactivate the microorganisms and enzymes in the suspension, thus obtaining tobacco stem fiber material. Step C: The tobacco stem fibers are mixed with tobacco dust and pulped, then paper-making, pressing and drying are performed to obtain a dried sheet base; The fermentation supernatant is concentrated to obtain a coating solution, which is then coated onto the dried tobacco substrate. After drying, the reconstituted tobacco leaves are obtained.

2. The method for preparing reconstituted tobacco leaves based on microbial fermentation according to claim 1, characterized in that, The microbial strain includes one or a combination of two of the following: Lactobacillus plantarum L8 strain or engineered Bacillus subtilis strain.

3. The method for preparing reconstituted tobacco leaves based on microbial fermentation according to claim 2, characterized in that, In step A The seed culture medium for the Lactobacillus plantarum L8 strain was MRS liquid medium, and the fermentation conditions were as follows: anaerobic conditions, temperature 30-45℃, pH 5.5-7.0, rotation speed 50-200 rpm, and culture time 24-72 h. The seed culture medium for the engineered Bacillus subtilis strain is LB liquid medium. The fermentation conditions are as follows: under aerobic conditions, solubility is greater than or equal to 30%, temperature is 30-45℃, pH value is 5.5-7.0, rotation speed is 50-200 rpm, and culture time is 24-72 h. The concentration of the inoculum solution is greater than or equal to 1×10⁻⁶. 8 CFU / mL.

4. The method for preparing reconstituted tobacco leaves based on microbial fermentation according to claim 3, characterized in that, In step B, The solid-liquid ratio of the inoculum to the tobacco stem material is 1:(0.1-0.2), 1:(0.2-0.5), 1:(0.5-0.8), 1:(0.8-1.2), 1:(1.2-2.0), or 1:(2.0-3.0), where the unit of solid is grams and the unit of liquid is milliliters.

5. The method for preparing reconstituted tobacco leaves based on microbial fermentation according to claim 4, characterized in that, The suspension contains tobacco stem matrix fibers, in-situ exfoliated cellulose nanofibers, and nanocrystals. The cellulose nanofibers have a diameter of 3-15 nm and a length of 1-8 μm; the nanocrystals are rod-shaped cellulose nanocrystals with a diameter of 5-20 nm and a length of 100-400 nm.

6. The method for preparing reconstituted tobacco leaves based on microbial fermentation according to claim 5, characterized in that, In step C The mass ratio of the tobacco stem fiber to the tobacco dust is (2-4):(6-8); The moisture content of the dried film substrate is 15-20%, and the quantitative content of the dried film substrate is 62±2 g / m³. 2 .

7. The method for preparing reconstituted tobacco leaves based on microbial fermentation according to claim 5, characterized in that, The coating solution is obtained by concentrating the fermentation supernatant to a solid content of 30-45%, and the coating amount of the coating solution is 30-38% of the mass of the dried substrate.

8. The method for preparing reconstituted tobacco leaves based on microbial fermentation according to claim 7, characterized in that, The reconstituted tobacco leaves exhibited inhibition rates of over 90% against Staphylococcus aureus, Escherichia coli, and Aspergillus niger.

9. A reconstituted tobacco leaf, characterized in that, The heated cigarette is prepared by the method for preparing reconstituted tobacco leaves based on microbial fermentation as described in any one of claims 1-8.

10. A heated cigarette, characterized in that, The heated cigarette comprises the reconstituted tobacco as described in claim 9.

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